The orphan 7-transmembrane segment receptor, Very Large G-protein coupled Receptor-1 (VLGR1, also termed Massl), is the largest known extracellular protein. Naturally occurring mutations in VLGR1 cause epilepsy in mice and humans, and Usher syndrome (sensorineural deafness and retinitis pigmentosa) in humans. The overall Aim of this project is to test the hypothesis that VLGR1 has essential functions in the retina and inner ear that cause Usher syndrome in mice as well as humans. Using in situ hybridization and immunohistochemistry, we will test the hypothesis that VLGR1 is expressed in the CNS, the retina and the inner ear of normal mice consistent with the pathogenesis of Usher syndrome. We will test the hypothesis that normal VLGR1 function requires the cytoplasmic and transmembrane domains, by comparing phenotypic effects of the naturally-occurring V2250X mutation and an engineered mutation that targets the G-protein proteolytic signal (GPS) and /-transmembrane segment (7-TM) domains while leaving the ectodomain intact (VLGR/del7TM). Using fundal photography, electroretinography, and light and electron microscopy, we will test the hypothesis that mutations of VLGR1 lead to visual system abnormalities in mutant mice. We will test the hypothesis that peripheral auditory deficits associated with the expression of non-functional forms of the VLGR1 protein reflect abnormal hair cell transduction. Functional studies will include auditory brainstem responses (ABR), distortion product otoacoustic emissions (DPOAEs) and endocochlear potentials. Cochlear morphology will be assessed by light and electron microscopy. We will test the hypothesis that VLGR1 forms a functional network with one or more proteins that are critical for hearing and vision, particularly proteins affected in other forms of Usher syndrome. To do so, we will co-transfect mammalian cells with appropriate VLGR1 expression constructs and constructs encoding other candidate proteins. We will determine which interactions are most likely to be biologically relevant by determining co-expression in mouse brain, eye and ear using in situ hybridization and/or immunohistochemistry. We will confirm these interactions in vivo by coimmunoprecipitation from mouse brains and/or retinas. To test the hypothesis that VLGR1 has functions that are partially complemented by other interacting proteins, particularly other Usher syndrome gene products, we will generate double mutant lines between VLGR1 and mice carrying mutations in the most biologically relevant interacting proteins. We are specifically interested in the other proteins with very large ectodomains, protocadherin- 15 and cadherin-23. These studies should shed new light on mechanisms controlling development of the retina and inner ear.